Mice were anesthetized with 1% sodium pentobarbital (50 mg/kg, i.p.) and transcardially perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4, 4°C) followed by 4% paraformaldehyde in PBS (pH 7.4, 4°C). DRGs (L4-5) were dissected from perfusion mice and postfixed in 4% paraformaldehyde in PBS for 30 minutes and cryoprotected in 30% sucrose at 4°C for 24 hours. DRGs were then embedded in an optimum cutting temperature compound (OCT, Leica, Wetalar, Germany) and rapidly frozen at −20°C (CM1950, Leica). Cryoembedded tissues were then cut into 20 μm thick slices using a sliding microtome (CM1950, Leica).
Sectioned DRGs were incubated in blocking solution (10% fetal bovine serum in PBS containing 0.1% Triton X-100) for 30 min at room temperature, followed by anti-TRPV1 (1 : 500; Neuromics, USA) at 4°C overnight. Afterwards, tissue sections were washed with 0.1% PBST and incubated in secondary antibody (1 : 200; Beyotime Biotechnology, China) at room temperature for 2 hours in the dark. Sections were then washed with 0.1% PBS and mounted with glycerol. All imaging was performed with an Olympus fluorescence microscope (BX51, Olympus Japan). Three mice from each group were analyzed. Quantitative analysis of immune response was consistent with previous studies [24 (link)].
Sectioned DRGs were incubated in blocking solution (10% fetal bovine serum in PBS containing 0.1% Triton X-100) for 30 min at room temperature, followed by anti-TRPV1 (1 : 500; Neuromics, USA) at 4°C overnight. Afterwards, tissue sections were washed with 0.1% PBST and incubated in secondary antibody (1 : 200; Beyotime Biotechnology, China) at room temperature for 2 hours in the dark. Sections were then washed with 0.1% PBS and mounted with glycerol. All imaging was performed with an Olympus fluorescence microscope (BX51, Olympus Japan). Three mice from each group were analyzed. Quantitative analysis of immune response was consistent with previous studies [24 (link)].